Structured illumination optical system and structured illumination microscope device

ABSTRACT

An illumination optical system includes: a beam splitter located near a conjugate position of a specimen and configured to split beams from a light source into a plurality of groups of beams having different splitting directions around an optical axis; a beam selector configured to select and transmit one group of beams from the plurality of groups of beams and that is rotatable with respect to the optical axis; and a ½ wavelength plate located near the beam selector and rotatable about the optical axis. The rotation angles of the ½ wavelength plate and of the beam selector about the optical axis are respectively set so that the polarization direction of the beam which has passed through the ½ wavelength plate is perpendicular to the splitting direction of the one group of beams that has been selected by the beam selector and split by the beam splitter.

This is a continuation of PCT International Application No.PCT/JP2012/004156, filed on Jun. 27, 2012, which is hereby incorporatedby reference. This application also claims the benefit of JapanesePatent Application No. 2011-143686, filed in Japan on Jun. 29, 2011,which is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a structured illumination opticalsystem and a structured illumination microscope device.

TECHNICAL BACKGROUND

Higher spatial resolution is demanded in the field of observation andmeasurement of a microstructure in a sample (specimen). As a method ofenhancing the spatial resolution, a technique to image a sample withperforming modulation using striped illumination light, and performingdemodulation the image by image processing has been proposed (e.g.Patent Documents 1 and 2).

The sixth embodiment disclosed in Patent Document 1 is an exampleapplied to a fluorescent observation device, and the optical systemthereof splits illumination light, including coherent light, emittedfrom a light source using a beam splitting element, such as adiffraction grating, then collects the plurality of split illuminationlight beams at a pupil position of an object lens, and allows the beamsto emit as parallel beams having different angles from the objectivelens, so as to form interference fringes overlapping in a neighborhoodof the observation object. By the illumination light modulated instripes, diffracted light, including spatial frequency components of theshape formation of the observation object, which cannot be transmittedby the imaging optical system alone, can be used for imaging. Then byrelatively modulating the phase of the split illumination light,interference fringes are moved on the observation object and a pluralityof images is acquired, so as to form an image by image processing.

In concrete terms, phase modulation to move the interference fringes isperformed by moving the beam splitting element in a directionperpendicular to the optical axis, or, according to another example, byinserting a wedge prism in one optical path of the illumination lightand moving the wedge prism in a direction perpendicular to the opticalaxis.

According to the method disclosed in Patent Document 2, illuminationlight including coherent light emitted from a light source is introducedvia an optical fiber, and is then split by a beam splitting unit, suchas a diffraction grating, and the plurality of split illumination lightis collected at a pupil position of an objective lens, so as to forminterference fringes in a neighborhood of the observation object. Justlike the above mentioned case, by the illumination light modulated instripes, high frequency components of the shape information of theobservation object, which cannot be transmitted by the imaging opticalsystem alone, can be used for imaging. Further, just like the abovementioned case, a plurality of images is acquired so as to form an imageby image processing.

In this method, in order to form one image, not only is a plurality ofimages acquired with modulating the phase of the illumination light, butalso the images are acquired with changing the orientation of theinterference fringes of the illumination light. This is because the highfrequency components can be used for forming an image only when thestructure has the same direction as the direction of the interferencefringes of the illumination light, therefore in order to reproduce theshape of the specimen that extends two-dimensionally, it is necessary toacquire and synthesize a plurality of images with changing the directionof the interference fringes.

In order to allow two or more beams to interfere in such a structuredillumination, it is normally preferable that the beams are S-polarizedlight with respect to the interference plane. This is because thecontrast when S-polarized light beams are interfered is 1, regardlessthe incident angles, but contrast when P-polarized light beams enterattenuates in proportion to cos (Δθ) with respect to a difference Δθ ofthe incident angles of the beams. The value of the contrast of theP-polarized light becomes negative when Δθ>90°, which means that thecontrast of the interference fringes is inverted.

Since a structured illumination microscope is a technique used forobtaining high resolution, it is desirable that the numerical aperture(NA) of the objective lens to be used is as large as possible and thefringe cycle of the structured illumination is as short as possible. Asa result, the beam for the structured illumination enters the specimenat a large angle, therefore if there is a P-polarized light component,attenuation of the P-polarized light is large because the abovementioned Δθ is large, which deteriorates the structured illuminationcontrast.

PRIOR ARTS LIST Patent Documents

-   Patent Document 1: U.S. Pat. No. 6,239,909(B1)-   Patent Document 2: U.S. Reissue Pat. No. 38307(E1)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

A conventional method of solving a problem of deterioration of thestructured illumination contrast is creating a non-polarized state by,for example, passing the light from the light source through a diffusionplate or oscillated multi-mode optical fiber, installing a polarizingplate, which rotates coaxially with the diffraction grating, in aneighborhood of the diffraction grating, and linearly polarizing thelight in the non-polarized state using the polarizing plate such thatthe light enters as S-polarized light on the specimen. As a result, goodstructured illumination contrast is obtained, but approximately ½ of thequantity of light is blocked by the polarizing plate, and the lightutilization efficiency becomes low, 50%.

With the foregoing in view, it is an object of the present invention toprovide a structured illumination optical system and a structuredillumination microscope that have a high efficiency to utilize the lightfrom the light source.

Means to Solve the Problems

A structured illumination optical system according to an aspect of thepresent invention includes: a beam splitter that is disposed near aconjugate position of a specimen and configured to split beams from alight source into a plurality of groups of beams having differentsplitting directions around a predetermined axis; and a beam selectorconfigured to select one group of beams from the plurality of groups ofbeams, the structured illumination optical system configured to form, onthe specimen, interference fringes by a plurality of beams included inthe one group of beams, and further including a ½ wavelength plate,wherein a fast axis of the ½ wavelength plate is set, based on the onegroup of beams selected by the beam selector, to a direction displacedfrom a predetermined direction by an angle θ/2, where θ is a differencebetween (i) a polarization direction of a beam that enters the ½wavelength plate and (ii) a polarization direction of a beam that is tobe emitted from the ½ wavelength plate.

A structured illumination optical system according to another aspect ofthe present invention includes: a beam splitter disposed near aconjugate position of a specimen and configured to split a beam from alight source into a plurality of beams; and a controller configured tocontrol a splitting direction of the plurality of beams, the structuredillumination optical system configured to form, on the specimen,interference fringes by the plurality of beams, and further including a½ wavelength plate, wherein a fast axis of the ½ wavelength plate isset, based on the splitting direction of the plurality of beams, to adirection displaced from a predetermined direction by an angle θ/2,where θ is a difference between (i) a polarization direction of a beamthat enters the ½ wavelength plate and (ii) a polarization direction ofa beam that is to be emitted from the ½ wavelength plate.

A structured illumination microscope according to an aspect of thepresent invention includes: a structured illumination optical systemaccording to any of the above aspects; a phase modulator configured tomodulate a phase of the plurality of beams; an image optical system thatforms an image of light from the specimen on an imaging plane of animaging device; and an image processing unit that generates a specimenimage by performing arithmetic processing on a plurality of imagesimaged by the imaging device each time the phase modulator modulates thephase of the plurality of beams.

Advantageous Effects of the Invention

According to the present invention, most of the light from the lightsource can be used as S-polarized structured illumination light, andlight utilization efficiency can be enhanced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting an overview of an optical system of astructured illumination microscope device according to Embodiment 1 ofthe present invention;

FIG. 2A is a schematic diagram depicting a configuration of adiffraction grating that is used for Embodiment 1, and FIG. 2B is aschematic diagram depicting a diffracted light split by the diffractiongrating;

FIGS. 3A and 3B are diagrams depicting a relationship between a ½wavelength plate that is used for Embodiment 1 and each rotation angleof a beam selection member (FIG. 3A is a state of being in a referenceposition, and FIG. 3B is a state of being rotated from the referenceposition);

FIGS. 4A, 4B, and 4C are diagrams depicting a relationship of a rotationangle of the beam selection member used for Embodiment 1 and the beamsto be selected (FIG. 4A is a case when the rotation angle of the beamselection member is θ₁ to the right, FIG. 4B is a case when the rotationangle of the beam selection member is θ₂ to the right, and FIG. 4C is acase when the rotation angle of the beam selection member is θ₃ to theleft);

FIG. 5 is a schematic diagram depicting a function of an illuminationoptical system according to Embodiment 1;

FIG. 6 is a diagram depicting a configuration of the beam selectionmember that is used for Embodiment 1;

FIGS. 7A and 7B are diagrams depicting a method of phase modulation bythe translational movement of the diffraction grating that is used forEmbodiment 1 (FIG. 7A is a general view, and FIG. 7B shows arelationship between a direction of each wave vector and a movingdistance);

FIG. 8 is a diagram depicting a configuration of a beam selection memberthat is used for a structured illumination microscope device accordingto Embodiment 2 of the present invention;

FIG. 9 is a diagram depicting a relationship of a rotation angle of thebeam selection member used for Embodiment 2 and beams to be selected;

FIG. 10 is a diagram depicting an overview of an optical system of astructured illumination microscope device according to Embodiment 3 ofthe present invention;

FIG. 11A is a schematic diagram depicting a configuration of adiffraction grating that is used for Embodiment 3, and FIG. 11B is aschematic diagram depicting a diffracted light split by the diffractiongrating;

FIGS. 12A and 12B are diagrams depicting a relationship between a ½wavelength plate that is used for Embodiment 3 and each rotation angleof the diffraction grating (FIG. 12A is a state of being in a referenceposition, and FIG. 12B is a state of being rotated from the referenceposition);

FIG. 13 is a schematic diagram depicting a function of an illuminationoptical system according to Embodiment 3; and

FIG. 14A is a schematic diagram depicting a configuration of liquidcrystal elements that are used for Embodiment 4, and FIG. 14B is aschematic diagram depicting a function as the ½ wavelength plate.

DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present invention will now be described withreference to the drawings. First, Embodiment 1 of the present inventionwill be described with reference to FIG. 1 to FIG. 7, and thenEmbodiment 2 of the present invention will be described mainly withreference to FIG. 8 and FIG. 9. Then Embodiment 3 of the presentinvention will be described with reference to FIG. 10 to FIG. 13.Finally Embodiment 4 of the present invention will be described withreference to FIG. 14.

Embodiment 1

As illustrated in FIG. 1, a structured illumination microscope device 1according to Embodiment 1 is used, for example, as a fluorescentobservation device that observes a specimen (sample) 5 havingfluorescent properties, and is constituted mainly by an objective lens6, a dichroic mirror 7, an illumination optical system (structuredillumination optical system according to this embodiment) that includesthe objective lens 6 and the dichroic mirror 7, an image optical system30 that also includes the objective lens 6 and the dichroic mirror 7, apicture element 35 (e.g. CCD, CMOS), an image storing and computingdevice 40, and an image display device 45.

The illumination optical system 10 includes, in order from the lightsource: an optical fiber 11, a collector lens 12, a diffraction grating13, a collective lens 16, a ½ wavelength plate 17, a beam selectionmember 18 (or beam selector), a lens 25, a field stop 26, a field lens27, an exciter filter 28, a dichroic mirror 7 and an objective lens 6.The image optical system 30 includes, in order from an object (specimen5): the objective lens 6, the dichroic mirror 7, a barrier filter 31 anda second objective lens 32. The image storing and computing device 40(or controller) is electrically connected with a translational drivingdevice 15 and a rotational driving device 24, which are described later,so as to control operation of the translational driving device 15 andthe rotational driving device 24, and constitute an image processingunit and a driving amount determining unit according to this embodiment.

The light from the light source (not illustrated) is guided by theoptical fiber 11, and the light emitted from the optical fiber 11 isconverted into parallel light by the collector lens 12. A laser light,for example, is used as the light source, since the illumination lightmust be coherent light. If the laser light is used, apolarization-preserving single mode fiber, for example, is used as theoptical fiber 11.

In this embodiment, the polarizing plate 23 is disposed between thecollector lens 12 and the diffraction grating 13. If the laser light isused as the light source, the polarization state thereof at the emittingend of the optical fiber 11 is approximately in a linearly polarizedstate, but it is preferable to pass the laser light through thepolarizing plate 23 so as to remove other polarization components. Theaxis of the polarizing plate 23 is matched with the polarization plane(direction of the oscillation axis of the electric field) if the emittedlight from the optical fiber 11 is polarized, but can be any directionif the emitted light from the optical fiber 11 is not polarized.

The diffraction grating 13 constitutes the beam splitting unit (or beamsplitter) according to this embodiment, and has a two-dimensional(triangular shaped) periodic structure spreading in a directionperpendicular to the optical axis of the illumination optical system 10,as illustrated in FIG. 2A. This periodic structure may be a periodicstructure formed by using density (transmittance) or a periodicstructure formed by using step difference (phase difference), but thephase difference type periodic structure is preferable in that thediffraction efficiency of the ±first order light is high.

The diffraction grating 13 splits the parallel light (coherent light)that enters from the collector lens 12 via the polarizing plate 23 intoa plurality of groups (three groups in this embodiment) of beams havingdifferent split directions (V₁ to V₃ indicated by dashed lines in FIG.2A) around the optical axis, and generates, in the beams of each group,a plurality of diffracted lights (e.g. 0 order diffracted light and±first order diffracted light) of which propagating directions(diffraction directions) are symmetric with respect to the optical axis.Each splitting direction V₁ to V₃ matches the direction of each wavevector of the diffraction grating 13 (direction perpendicular to thegrid line (marked line) of the diffraction grating 13).

Each of the diffracted lights generated by the diffraction grating 13 iscollected at a position within a pupil conjugate plane by the collectivelens 16 illustrated in FIG. 1, according to the diffraction directionand the diffraction order of each diffracted light. For example, asillustrated in FIG. 2B, the 0 order diffracted light 14 a is collectedat the center of the pupil conjugate plane (position of the opticalaxis), and three +first order diffracted lights 14 b, 14 c and 14 d andthree −first order diffracted lights 14 e, 14 f and 14 g are collectedrespectively with equal angle intervals (60° intervals in thisembodiment) on a circle of which center is the 0 order diffracted light14 a (position of the optical axis). The 0 order diffracted light 14 aand the ±first order diffracted lights 14 g and 14 d constitute a groupof beams along the splitting direction V₁ (hereafter called “first groupof beams”), the 0 order diffracted light 14 a and the ±first orderdiffracted lights 14 c and 14 f constitute a group of beams along thesplitting direction V₂ (hereafter called “second group of beams”), andthe 0 order diffracted light 14 a and ±first order diffracted lights 14b and 14 e constitute a group of beams along the splitting direction V₃(hereafter called “third group of beams”).

As illustrated in FIG. 1, the diffraction grating 13 is constructed sothat translational movement in a direction perpendicular to the opticalaxis of the illumination optical system 10 is possible by thetranslational driving device 15, such as a piezo motor (constitutes thephase modulation unit in this embodiment), and the phase of theillumination light is modulated by the translational movement (describedin detail later).

The ½ wavelength plate 17 (see FIG. 1) transforms the polarizationdirections of the plurality of diffracted light (0 order diffractedlight and ±first order diffracted lights) generated by the diffractiongrating 13 into a predetermined direction of linearly polarized lightrespectively. The direction of the fast axis of the ½ wavelength plate17 is set to a direction to be the S-polarized light when apredetermined group of beams (one of the first group of beams to thethird group of beams), that is selected by the beam selection member 18after passing through the ½ wavelength plate 17, is irradiated onto thespecimen 5, specifically to a direction that is perpendicular to thesplitting direction of a predetermined one group of beams (one of thethree splitting directions, V₁ to V₃, indicated by dashed lines in FIG.2A) selected by the beam selection member 18 (that is, a direction inwhich the polarization direction of the predetermined one group of beamsthat is selected is parallel with the tangential line of a circle whichcontacts the beams and of which center is the optical axis. The fastaxis of the ½ wavelength plate refers to a direction of polarization inwhich phase delay is smallest when the polarized light passed throughthe ½ wavelength plate (this definition is the same hereinbelow).

As shown in FIG. 4 and FIG. 6, the beam selection member 18 is a diskhaving first and second apertures 19 and 20 where ±first orderdiffracted lights (linearly polarized lights) generated by the ½wavelength plate 17 can pass through respectively, and is disposed nearthe pupil conjugate plane where the diffracted light is collected by thecollective lens 16 (see FIG. 1). The first aperture 19 and the secondaperture 20 are formed symmetrically with respect to the center of thebeam selection member 18, so that only a pair of ±first order diffractedlights (±first order diffracted lights that belong to one group ofbeams, out of the first group of beams to the third group of beams)disposed in symmetrical positions with respect to the optical axis ofthe illumination optical system 10 (that is, the propagating directions(diffraction directions) are symmetric with respect to the opticalaxis), out of the plurality of ±first order diffracted lights collectedon the pupil conjugate plane (six ±first order diffracted lights 14 b to14 g shown in FIG. 2B), can pass through the first aperture 19 and thesecond aperture 20.

In concrete terms, in FIG. 2B, the beam selection member 18 selects oneof the pairs of the ±first order diffracted lights 14 g and 14 d(belonging to the first group of beams) which are vertically symmetricwith respect to the optical axis of the illumination optical system 10,the ±first order diffracted lights 14 c and 14 f (belonging to thesecond group of beams) which are symmetric inclining 120° to the rightviewed from the microscope (60° to the left), and the ±first orderdiffracted lights 14 b and 14 e (belonging to the third group of beams)which are symmetric inclining 120° to the left viewed from themicroscope (60° to the right), and the selected pair passes through thefirst aperture 19 and the second aperture 20.

As illustrated in FIG. 1, the beam selection member 18 is constructed tobe rotatable, together with the ½ wavelength plate 17 disposed near thebeam selection member 18, around the center of the beam selection member18 (optical axis of the illumination optical system 10) as a rotationaxis, by the rotational driving device 24, which is an electric motor,for example, and by the rotation of the beam selection member 18, onepair of ±first order diffracted lights, out of the three pairs of ±firstdiffracted lights disposed symmetrically with respect to the opticalaxis of the illumination optical system 10, can be selected. In otherwords, diffracted light (±first order diffracted light) having desireddiffraction direction and diffraction order can be selected by rotatingthe beam selection member 18, whereby the structured illuminationdirection can be selected.

On the other hand, the beam selection member 18 also has a function tocut off diffracted light of an unnecessary order (e.g. 0 orderdiffracted light 14 a) out of the diffracted lights generated by thediffraction grating 13. In other words, in this embodiment, only ±firstorder diffracted lights are used as the illumination light, and 0 orderdiffracted light and diffracted lights of the second or higher order arecut off by the beam selection member 18. The 0 order diffracted lightindicated by the solid line in FIG. 1 therefore does not actually existafter the beam selection member 18, but is illustrated for convenienceto clearly show the paths of the beams.

The rotation angles of the ½ wavelength plate 17 and the beam selectionmember 18 are controlled as follows. As illustrated in FIG. 3A, thereference position of the ½ wavelength plate 17 is a position where thedirection of the fast axis thereof is parallel with the direction of theaxis of the polarizing plate 23 (hereafter called “first referenceposition”), and the reference position of the beam selection member 18is a position where the beam selection direction of the beam selectionmember 18 (direction of the line connecting the center of the firstaperture 19 and the center of the second aperture 20) is perpendicularto the direction of the axis of the polarizing plate 23 (hereaftercalled “second reference position”).

As illustrated in FIG. 3B, the ½ wavelength plate 17 is constructed soas to rotate in the same direction as the rotation of the beam selectionmember 18 at ½ rotation speed thereof. In concrete terms, when thedirection of the fast axis of the ½ wavelength plate 17 rotates to theright (viewed from the microscope, this definition is the samehereinbelow) from the first reference position by the rotation angleθ/2, the beam selection direction of the beam selection member 18rotates to the right from the second reference position by the rotationangle θ. This mechanism for rotating the ½ wavelength plate 17 at therotation speed that is ½ of the rotation speed of the beam selectionmember 18 can be Implemented by setting the gear ratio of each gear ofthe ½ wavelength plate 17 and the beam selection member 18, which engagewith the rotational driving axis of the single rotational driving device24 at 2:1.

If the beam selection direction of the beam selection member 18 isrotated to the right from the second reference position by the rotationangle θ₁ using this mechanism, as illustrated in FIG. 4A, so as toselect a pair of ±first order diffracted lights 14 g and 14 d whichbelong to the first group of beams, out of the three pairs of ±firstorder diffracted lights, the direction of the fast axis of the ½wavelength plate 17 rotates to the right from the first referenceposition by the rotation angle θ₁/2. While the polarization direction(indicated by a broken line with bidirectional arrows) of each ±firstorder diffracted light before passing through the ½ wavelength plate 17is parallel with the direction of the axis of the polarizing plate 23 atthis time, the polarization direction of each ±first order diffractedlight after passing through the ½ wavelength plate 17 rotates to theright by the rotation angle θ₁ (in FIG. 4A, only the polarizationdirections of the pair of ±first order diffracted lights 14 g and 14 dselected by the beam selection member 18 are indicated by a solid linewith bidirectional arrows), and is perpendicular to the beam selectiondirection of the beam selection member 18 (matches with the splittingdirection V₁ indicated in FIG. 2A).

In the same manner, if the beam selection direction of the beamselection member 18 is rotated to the right from the second referenceposition by the rotation angle θ₂, as illustrated in FIG. 4B, so as toselect a pair of ±first order diffracted lights 14 c and 14 f whichbelong to the second group of beams, out of the three pairs of ±firstorder diffracted lights, the direction of the fast axis of the ½wavelength plate 17 rotates to the right from the first referenceposition by the rotation angle θ₂/2. While the polarization direction(indicated by a broken line with bidirectional arrows) of each ±firstorder diffracted light before passing through the ½ wavelength plate 17is parallel with the direction of the axis of the polarizing plate 23 atthis time, the polarization direction of each ±first order diffractedlight after passing through the ½ wavelength plate 17 rotates to theright by the rotation angle θ₂ (in FIG. 4B, only the polarizationdirections of the pair of ±first order diffracted lights 14 c and 14 fselected by the beam selection member 18 are indicated by a solid linewith bidirectional arrows), and is perpendicular to the beam selectiondirection of the beam selection member 18 (matches with the splittingdirection V₂ indicated in FIG. 2A).

If the beam selection direction of the beam selection member 18 isrotated to the left (viewed from the microscope, this definition is thesame hereinbelow) from the second reference position by the rotationangle θ₃ as illustrated in FIG. 4C, so as to select a pair of ±firstorder diffracted lights 14 b and 14 e which belong to the third group ofbeams, out of the three pairs of ±first order diffracted lights, thedirection of the fast axis of the ½ wavelength plate 17 rotates to theleft from the first reference position by the rotation angle θ₃/2. Whilethe polarization direction (indicated by a broken line withbidirectional arrows) of each ±first order diffracted light beforepassing through the ½ wavelength plate 17 is parallel with the directionof the axis of the polarizing plate 23 at this time, the polarizationdirection of each ±first order diffracted light after passing throughthe ½ wavelength plate 17 rotates to the left by the rotation angle θ₃(in FIG. 4C, only the polarization directions of the pair of ±firstorder diffracted lights 14 b and 14 e selected by the beam selectionmember 18 are indicated by a solid line with bidirectional arrows), andis perpendicular to the beam selection direction of the beam selectionmember 18 (matches with the splitting direction V₃ indicated in FIG.2A).

By rotating the ½ wavelength plate 17 in the same direction as therotation of the beam selection member 18 at a half of the rotation speedthereof like this, a pair of ±first order diffracted lights selected bythe beam selection member 18 can always be in a polarization statehaving a field oscillation direction that is perpendicular to thesplitting direction (diffraction direction) of the selected pair of±first order diffracted lights (parallel with the rotation direction ofthe beam selection member 18), therefore all the ±first order diffractedlights selected by the beam selection member 18 can be S-polarized onthe irradiation surface. FIG. 5 is a schematic diagram depicting thefunction of the illumination optical system 10 (particularly thediffraction grating 13, the ½ wavelength plate 17, and the beamselection member 18) described above. In FIG. 5, a bidirectional arrowenclosed by a circle indicates the polarization direction of the beam,and a bidirectional arrow enclosed by a square indicates the axialdirection of the optical element.

As illustrated in FIG. 6, a plurality of (six in the case of FIG. 6)notches 21, 21, - - - are formed on the periphery of the beam selectionmember 18, and the rotation angle of the beam selection member 18 can bedetected by the timing sensor 22 detecting a notch 21.

As described above, the diffracted light (±first order diffractedlights), of which diffraction direction and diffraction order areselected by the beam selection member 18 and of which polarization stateis controlled by the ½ wavelength plate 17 forms a conjugate surfacewith the diffraction grating 13 near the field stop 26, by the lens 25,is converted into parallel light by the field lens 27, reflected by thedichroic mirror 7 via the exciter filter 28, and is collected on thepupil plane 6 a of the objective lens 6 again for each diffractionorder, as illustrated in FIG. 1. Each beam (diffracted light) emittedfrom the objective lens becomes a parallel beam as the illuminationlight, and is irradiated onto the specimen 5. In this case, each beam(diffracted light) is coherent light, therefore it irradiates thespecimen 5 as light having the structure of interference fringes withequal intervals. The illumination light having a fringe structure iscalled “structured illumination light”, and this illumination method iscalled “structured illumination method”.

If the specimen 5 is illuminated by the structured illumination light,moire interference fringes are generated by the interference between theperiodic structure of the illumination light and the periodic structureof the specimen 5, but these moire interference fringes can enter theobjective lens 6, because moire interference fringes have a lowerfrequency than the original frequency even if high frequency shapeinformation of the specimen 5 is included.

The light (fluorescence), which is generated from the surface of thespecimen 5 and enters the objective lens 6, is converted into parallellight by the objective lens 6, is transmitted through the dichroicmirror 7 and the barrier filter 31, and forms an image of the specimenon the imaging plane 36 of the picture element 35 by the secondobjective lens 32.

The picture element 35 captures an image formed by the image opticalsystem 30, and outputs image signals to the image storing and computingdevice 40. The image captured by the picture element 35 is an imagegenerated by being illuminated by spatially modulated illumination light(structure illumination light), hence a super resolution image of thespecimen 5 (specimen image) is generated by restoring the image by theimage storing and computing device 40 using inverse modulation based onknown predetermined image processing. For example, a method disclosed inU.S. Pat. No. 8,115,806 can be used. The super resolution image of thespecimen 5 (specimen image) generated by the image storing and computingdevice 40 is stored in an internal memory (not illustrated), and isdisplayed on the image display device 45.

To restore the original image by image processing, the same specimen 5may be imaged with modulating the phase of the interference fringes byillumination three or more times. Because a modulated image includesthree unknown parameters (0 order component, +first order component, and−first order component) in the optical information which is obtainedwhen the frequency component of the specimen is diffracted by thestructured illumination, and in order to determine the unknownparameters by arithmetic processing, a plurality of images exceeding thenumber of the unknown parameters are required.

According to this embodiment, in order to modulate the phase of theinterference fringes generated by the illumination light, thediffraction grating 13 is driven in a direction perpendicular to theoptical axis of the illumination optical system 10 (x direction in FIG.7A) using the translational driving device 15 as illustrated in FIG. 7A.In this case, the driving amount of the diffraction grating 13 isdifferent depending on the direction of the diffracted light, which isselected by the beam selection member 18. In concrete terms, when Pdenotes the cycle (pitch) of the diffraction grating 13, and θ denotesan angle formed by the direction of the wave vector of the diffractiongrating 13 (three directions indicated by the dashed lines in thedrawing) and the driving direction of the diffraction grating 13 by thetranslational driving device 15 (θ₁ to θ₃ in FIG. 7B corresponds to θ₁to θ₃ in FIG. 4) as illustrated in FIG. 7B, the distance L to drive thediffraction grating 13 for the phase modulation amount of the structuredillumination (interference fringes generated by the illumination light)to be a predetermined value φ is given by the following Expression 1.

L=φ×P/(4π×|cos θ|)  (1)

To establish φ=2π, L=P/(2×|cos θ|), which appears to be using only ahalf cycle of the diffraction grating 13, but this is because thestructured illumination in this embodiment utilizes the interference of±first order diffracted lights.

In this embodiment, in order to acquire two-dimensional super resolutionimage data, ±first order diffracted lights are generated in threedirections of which inclinations are 120° different from each other, bythe diffraction grating 13, as mentioned above, and images on the ±firstdiffracted lights in three directions are acquired by selecting ±firstorder diffracted lights in one direction at a time out of the threedirections, by rotating the beam selection member 18. In this case, eachphase-modulated image can be acquired according to the followingsequence. In the following description, it is assumed that the phasemodulation of the illumination light (interference fringes) is performedthree times for the illumination light (±first order diffracted lights)in each direction.

First as illustrated in FIG. 4A, the beam selection direction of thebeam selection member 18 is stopped at a position that is rotated to theright from the second reference position by the rotation angle θ₁, and apair of ±first order diffracted lights 14 g and 14 d is selected as theillumination light. In this case, the fast axis of the ½ wavelengthplate 17 stops at a position that is rotated to the right from the firstreference position by the rotation angle θ₁/2. During this time, thediffraction grating 13 is intermittently or continuously moved by thetranslational driving device 15 in a direction (direction of the angleθ₁ in FIG. 7B) that corresponds to the diffraction direction of the±first order diffracted lights 14 g and 14 d (corresponds to thesplitting direction V₁ indicated in FIG. 2A), so that the first phaseimage is acquired at the first movement position in this direction, thenthe second phase image is acquired at the second movement position, andfinally the third phase image is acquired at the third movementposition.

Then, as illustrated in FIG. 4B, the beam selection direction of thebeam selection member 18 is stopped at a position that is rotated to theright from the second reference position by the rotation angle θ₂, and apair of ±first order diffracted lights 14 c and 14 f is selected as theillumination light. In this case, the fast axis of the ½ wavelengthplate 17 stops at a position that is rotated to the right from the firstreference position by the rotation angle θ₂/2. During this time, thediffraction grating 13 is intermittently or continuously moved by thetranslational driving device 15 in a direction (direction of the angleθ₂ in FIG. 7B) that corresponds to the diffraction direction of the±first order diffracted lights 14 c and 14 f (corresponds to thesplitting direction V₂ indicated in FIG. 2A), so that the first phaseimage is acquired at the first movement position in this direction, thenthe second phase image is acquired at the second movement position, andfinally the third phase image is acquired at the third movementposition.

Then as illustrated in FIG. 4C, the beam selection direction of the beamselection member 18 is stopped at a position that is rotated to the leftfrom the second reference position by the rotation angle θ₃, and a pairof ±first order diffracted lights 14 b and 14 e is selected as theillumination light. In this case, the fast axis of the ½ wavelengthplate 17 stops at a position that is rotated to the left from the firstreference position by the rotation angle θ₃/2. During this time, thediffraction grating 13 is intermittently or continuously moved by thetranslational driving device 15 in a direction (direction of the angleθ₃ in FIG. 7B) that corresponds to the diffraction direction of the±first order diffracted lights 14 b and 14 e (corresponds to thesplitting direction V₃ indicated in FIG. 2A), so that the first phaseimage is acquired at the first movement position in this direction, thenthe second phase image is acquired at the second movement position, andfinally the third phase image is acquired at the third movementposition.

In this embodiment, the ½ wavelength plate 17 is rotated in the samedirection as the rotation of the beam selection member 18 at ½ rotationspeed thereof, whereby the illumination light (a pair of ±first orderdiffracted lights) selected by the beam selection member 18 enters thespecimen 5 as S-polarized light, but the present invention is notlimited to this mode, and modes of rotation of the ½ wavelength plate 17and the beam selection member 18 can be freely set, e.g. rotating the ½wavelength plate 17 and the beam selection member 18 in oppositedirections, only if the illumination light selected by the beamselection member 18 results in S-polarized light. In concrete terms, inthe case of FIG. 4A, the beam selection direction of the beam selectionmember 18 is in a position that is rotated to the right from the secondreference position by the rotation angle θ₁, and if the pair of ±firstorder diffracted lights 14 g and 14 d is selected as the illuminationlight, the position of the fast axis of the ½ wavelength plate 17, togenerate a polarization state in which the pair of ±first orderdiffracted lights 14 g and 14 d is irradiated onto the specimen 5 as theS-polarized light, is not limited to the position that is rotated to theright from the first reference position by the rotation axis θ₁/2.

In other words, the position that is rotated from this position by ±90°(that is, the position that is rotated to the left from the firstreference position by 90°−θ₁/2, which results in the same position asthe position that is rotated to the right from the first referenceposition by θ₁/2+90°) is also a position where the same polarizationstate is generated. Therefore in the case of rotating the beam selectiondirection of the beam selection member 18 to the right from the secondreference position by the rotation axis θ₁, the same functional effectcan be implemented by rotating the fast axis of the ½ wavelength plate17 to the left from the first reference position by 90°−θ₁/2, forexample, hence this latter mode may be used instead. The first referenceposition and the second reference position mentioned above are notlimited to the positions indicated in FIG. 4, but may be set at anyappropriate positions.

Embodiment 2

A structured illumination microscope device according to Embodiment 2 ofthe present invention is the structured illumination microscope device 1according to Embodiment 1 depicted in FIG. 1, where the beam selectionmember 18A illustrated in FIG. 8 is used instead of the beam selectionmember 18, and the other configuration is the same as Embodiment 1. Thefunctional aspects are also the same as Embodiment 1, except for thedifference generated by using the beam selection member 18A. Hence inthe description in Embodiment 2, the drawings used for describingEmbodiment 1, including FIG. 1, are referred to when needed, anddescription on matters the same as Embodiment 1 is omitted.

The only difference of the beam selection member 18A illustrated in FIG.8 from the beam selection member 18 of Embodiment 1 illustrated in FIG.6 is that there is a third aperture 29 at the center, and the rest ofthe configuration is the same as the beam selection member 18. Thereforein the beam selection member 18A illustrated in FIG. 8, a composingelement the same as the beam selection member 18 illustrated in FIG. 6is denoted with a same reference symbol as used in FIG. 6, for whichdetailed description is omitted.

The third aperture 29 of the beam selection member 18A illustrated inFIG. 8 is configured such that the 0 order diffracted light 14 a (seeFIG. 2B), which is cut off in the beam selection member 18 illustratedin FIG. 6, is allowed to pass through. In other words, the structuredillumination microscope device of Embodiment 2, which uses the beamselection member 18A, uses the 0 order diffracted light 14 a that passesthrough the third aperture 29 along with the pair of ±first orderdiffracted light that passes through the first aperture 19 and thesecond aperture 20 (one of the pairs of 14 g and 14 d, 14 c and 14 f,and 14 b and 14 e as illustrated in FIG. 2B), and this is the majordifference from the structured illumination microscope device ofEmbodiment 1.

In concrete terms, if the 0 order diffracted light 14 a and a pair of±first order diffracted lights 14 g and 14 d (first group of beams) areselected as the illumination light, for example, the beam selectiondirection of the beam selection member 18A is stopped at a position thatis rotated to the right from the second reference position by therotation axis θ₁, as illustrated in FIG. 9. In this case, the fast axisof the ½ wavelength plate 17 (see FIG. 5) stops at a position that isrotated to the right from the first reference position by the rotationangle θ₁/2.

In this state, the 0 order diffracted light 14 a and the pair of ±firstorder diffracted lights 14 g and 14 d are selected by the beam selectionmember 18A as the illumination light, and the selected diffracted lights14 a, 14 g and 14 d are polarized by the ½ wavelength plate 17 to beS-polarized light when irradiated onto the specimen 5 (see FIG. 1). Ifthe 0 order diffracted light 14 a and the pair of ±first orderdiffracted lights 14 c and 14 f (second group of beams) are selected asthe illumination light, the beam selection member 18A is set in the samestate as the beam selection member 18 illustrated in FIG. 4B, and if the0 order diffracted light 14 a and the pair of ±first order diffractedlights 14 b and 14 e (third group of beams) are selected as theillumination light, the beam selection member 18A is set in the samestate as the beam selection member 18 illustrated in FIG. 4C.

If beams of each group are selected as the illumination light, thediffraction grating 13 is moved to modulate the phase of theillumination light, and an image of the specimen 5 is acquired each timethe phase is modulated, just like Embodiment 1. A number of times ofperforming the phase modulation can be three, as in the case ofEmbodiment 1, but the number may be different. For example, the phasemodulation may be performed five times at each of the three selectedpositions, whereby a total of 15 images, five phases in threedirections, can be acquired.

Embodiment 3

As illustrated in FIG. 10, a structured illumination microscope device51 according to Embodiment 3 is used for similar purposes as Embodiment1 and Embodiment 2 described above, and is constituted mainly by anobjective lens 56, a dichroic mirror 57, an illumination optical system60 (structured illumination optical system according to this embodiment)that includes the objective lens 56 and the dichroic mirror 57, an imageoptical system 80 that also includes the objective lens 56 and thedichroic mirror 57, a picture element 85 (e.g. CCD and CMOS), an imagestoring and computing device 90, and an image display device 95.

The illumination optical system 60 includes, in order from the lightsource: an optical fiber 61, a collector lens 62, a ½ wavelength plate63, a diffraction grating 64, a collective lens 66, a light shieldingplate 67, a phase plate 68, a lens 75, a field stop 76, a field lens 77,an exciter filter 78, a dichroic mirror 57, and an objective lens 56.The image optical system 80 includes, in order from an object (specimen55): the objective lens 56, the dichroic mirror 57, a barrier filter 81,and a second objective lens 82. The image storing and computing device90 (or controller) is electrically connected with rotational drivingdevices 72 and 73, which are described later, so as to control operationof the rotational driving devices 72 and 73, and constitute an imageprocessing unit according to this embodiment.

The light from the light source (not illustrated) is guided by theoptical fiber 61, and the light emitted from the optical fiber 61 istransformed into parallel light by the collector lens 62. A laser light,for example, is used as the light source, since the illumination lightmust be coherent light. If a laser light is used, apolarization-preserving single mode fiber, for example, is used as theoptical fiber 61.

In this embodiment, a polarizing plate 71 is disposed between thecollector lens 62 and the ½ wavelength plate 63. If a laser light isused as the light source, the polarization state thereof at the emittingend of the optical fiber 61 is approximately in a linearly polarizedstate, but it is preferable to pass the laser light through thepolarizing plate 71 so as to remove the other polarization components.The axis of the polarizing plate 71 is matched with the polarizationplane (direction of the oscillation axis of the electric field) if theemitted light from the optical fiber 61 is polarized, but can be anydirection if the emitted light from the optical fiber 61 is notpolarized.

The ½ wavelength plate 63 transforms the polarization direction of theparallel light (coherent light) that enters from the collector lens 62via the polarizing plate 71 into a predetermined direction of linearlypolarized light. The direction of the fast axis of the ½ wavelengthplate 63 is set to a direction to be the S-polarized light when aplurality of beams (0 order diffracted light 65 a and ±first orderdiffracted lights 65 b and 65 c (see FIG. 11B) described later) split bythe diffraction grating 64 is irradiated onto the specimen 55 afterpassing through the ½ wavelength plate 63, specifically to a directionthat is perpendicular to the beam splitting direction W (indicated bythe dashed line in FIG. 11A) of the diffraction grating 64 to bedescribed below (a direction parallel with the grid line (marked line)of the diffraction grating 64).

The diffraction grating 64 constitutes the beam splitting unit (or beamsplitter) according to this embodiment, and has a one-dimensionalperiodic structure in a direction perpendicular to the optical axis ofthe illumination optical system 10, as illustrated in FIG. 11A. Thisperiodic structure may be a periodic structure formed by using density(transmittance), or a periodic structure formed by using a stepdifference (phase difference), but the phase difference type periodicstructure is preferable in that the diffraction efficiency of the ±firstorder light is high.

The diffraction grating 64 splits the linearly polarized light thatenters from the ½ wavelength plate 63 into a plurality of beams alongthe beam splitting direction W (see FIG. 11A) of the diffraction grating64, and generates a plurality of diffracted light (e.g. 0 orderdiffracted light 65 a and ±first order diffracted lights 65 b and 65 cillustrated in FIG. 11B), of which propagating directions (diffractiondirections) are symmetric with respect to the optical axis. The beamsplitting direction W matches the directions of the wave vector of thediffraction grating 64 (direction perpendicular to the grid line (markedline) of the diffraction grating 13).

Each of the plurality of diffracted lights generated by the diffractiongrating 64 is collected at a position within a pupil conjugate plane bythe collective lens 66 illustrated in FIG. 10, according to thediffraction direction and the diffraction order of each diffractedlight. For example, as illustrated in FIG. 11B, the 0 order diffractedlight 65 a is collected at the center of the pupil conjugate plane(position of the optical axis), and the pair of ±first order diffractedlights 65 b and 65 c are collected at symmetric positions with respectto the 0 order diffracted light 65 a (position of the optical axis).

As illustrated in FIG. 10, the diffraction grating 64 is constructed sothat rotational driving device 72, e.g. electric motor, can rotate thediffraction grating 64 together with the ½ wavelength plate 63 disposednear the diffraction grating 64, around the center of the diffractiongrating 64 (optical axis of the illumination optical system 60) as therotation axis. By rotating the ½ wavelength plate 63 and the diffractiongrating 64, a diffracted light having a desired diffraction directionand a diffraction order (±first order diffracted light) can be used asthe illumination light, and as a result, the structured illuminationdirection can be selected.

The rotation angles of the ½ wavelength plate 63 and the diffractiongrating 64 are controlled as follows. As illustrated in FIG. 12A, thereference position of the ½ wavelength plate 63 is a position where thedirection of the fast axis thereof is parallel with the direction of theaxis of the polarizing plate 71 (hereafter called “third referenceposition”), and the reference position of the diffraction grating 64 isa position where the beam splitting direction of the diffraction grating64 is perpendicular to the direction of the axis of the polarizing plate71 (hereafter called “fourth reference position”).

As illustrated in FIG. 12B, the ½ wavelength plate 63 is constructed soas to rotate in the same direction as the rotation of the diffractiongrating 64 at ½ rotation speed thereof. In concrete terms, when thedirection of the fast axis of the ½ wavelength plate 63 rotates to theright (viewed from the microscope, this definition is the samehereinbelow) from the third reference position by the rotation angleα/2, the beam splitting direction of the diffraction grating 64 rotatesto the right from the fourth reference position by the rotation angle α.This mechanism for rotating the ½ wavelength plate 17 at a rotationspeed that is ½ of the rotation speed of the diffraction grating 64 canbe implemented by setting the gear ratio of each gear of the ½wavelength plate 63 and the diffraction grating 64, which engage withthe rotational driving axis of the rotational driving device 72, at 2:1.

By rotating the ½ wavelength plate 63 in the same direction as therotation of the diffraction grating 64 at half of the rotation speedthereof like this, the diffracted light split by the diffraction grating64 can always be in a polarized state having a field oscillationdirection that is perpendicular to the beam splitting direction(diffraction direction) of the diffraction grating 64 (parallel with therotation direction of the diffraction grating 64), therefore all thediffracted light that passes through the diffraction grating 64 can beS-polarized light emitted onto the irradiation surface. FIG. 13 is aschematic diagram depicting the function of the illumination opticalsystem 60 (particularly the ½ wavelength plate 17 and the diffractiongrating 64) described above. In FIG. 13, a bidirectional arrow enclosedby a circle indicates the polarization direction of the beam, and abidirectional arrow enclosed by a square indicates the axial directionof the optical element.

As described above, the diffracted light, of which polarization state iscontrolled by the ½ wavelength plate 63 and of which diffractiondirection and diffraction order are controlled by the diffractiongrating 64, forms a conjugate surface with the diffraction grating 64near the light shielding plate 67 by the lens 66, where 0 orderdiffracted light and second or higher order diffracted lights are cutoff by the light shielding plate 67, and only the ±first orderdiffracted lights enter the phase plate 68 as illustrated in FIG. 10.Since only the ±first order diffracted lights are used as theillumination light in this embodiment, the 0 order diffracted lightindicated by the solid line in FIG. 10 does not actually exist after thelight shielding plate 67, but is illustrated out of convenience, justlike the case of Embodiment 1. The 0 order diffracted light may be usedas the illumination light, just like Embodiment 2.

The phase plate 68 has a thickness of a transparent substrate or has adeposited thin film of which film thickness is controlled, so that anappropriate phase delay is generated on one or both of the enteredbeam(s) (±first order diffracted light(s)). Thereby a phase differenceis generated between the +first order diffracted light and the −firstorder diffracted light. The phase difference can be modulated by therotational driving device 73 rotating the phase plate 68 around theoptical axis, changing the positions where the ±first order diffractedlights passes through the phase plate 68.

The beams (±first order diffracted light), of which phases are modulatedby the phase plate 68 form a conjugate surface with the diffractiongrating 64 near the field stop 76 by the lens 75, are converted intoparallel light by the field lens 77, reflected by the dichroic mirror 57via the exciter filter 78, and are collected on the pupil plane 56 a ofthe objective lens 56 again for each diffraction order. Each beam(diffracted light) emitted from the objective lens becomes a parallelbeam as the illumination light, and is irradiated onto the specimen 55.In this case, each beam (diffracted light) is coherent light, thereforeeach beam is irradiated onto the specimen 55 as the structuredillumination light having a structure of interference fringes with equalintervals.

If the specimen 55 is illuminated by the structured illumination light,moire interference fringes are generated by the interference between theperiodic structure of the illumination light and the periodic structureof the specimen 55, but the moire interference fringes can enter theobjective lens 56, because moire interference fringes have a lowerfrequency than the original frequency, even if high frequency shapeinformation of the specimen 55 is included.

The light (fluorescence), which is generated from the surface of thespecimen 55 and enters the objective lens 56, is converted into parallellight by the objective lens 56, is transmitted through the dichroicmirror 57 and the barrier filter 81, and forms an image of the specimenon the imaging plane 86 of the picture element 85 by the secondobjective lens 82.

The picture element 85 captures an image formed by the image opticalsystem 80, and outputs image signals to the image storing and computingdevice 90. The image captured by the picture element 85 is an imagegenerated by being illuminated by the spatially modulated illuminationlight (structured illumination light), hence a super resolution image ofthe specimen 55 (specimen image) is generated by restoring the image bythe image storing and computing device 90 using inverse modulation basedon a known predetermined image processing. The super resolution image ofthe specimen 55 (specimen image) generated by the image storing andcomputing device 90 is stored in an internal memory (not illustrated),and is displayed on the image display device 95.

To restore the original image by image processing, the same specimen 55may be imaged with modulating the phase of the interference fringes byillumination light three or more times due to the same reason asdescribed in Embodiment 1. In this embodiment, the phase plate 68 isrotated by the rotational driving device 73 to modulate the phase of theinterference fringes by the illumination light, as described above, butthe phase modulation may be performed by driving the diffraction grating64 translationally in the beam splitting direction (direction in whichdiffraction is generated). In this case, the phase plate 68 is notnecessary.

In this embodiment, where the diffraction grating 64 rotates, thestructured illumination light (±first order diffracted light) can beirradiated onto the specimen 55 in any direction around the optical axisof the objective lens 56 in order to acquire two-dimensional superresolution image data, and in concrete terms, the structuredillumination light can be irradiated from three directions, of whichinclination angles are different by 120° from each other, by rotatingthe diffraction grating 64 such that the beam splitting directionthereof changes 120° each time, for example. In this case, the phasemodulation of the structured illumination light is performed four timesat each rotation position of the diffraction grating 64, whereby a totalof twelve images, four phases in three directions, can be acquired.

In this embodiment, the ½ wavelength plate 63 is rotated in the samedirection as the rotation of the diffraction grating 64 at ½ rotationspeed thereof, whereby the illumination light (a pair of ±first orderdiffracted light) generated by the diffraction grating 64 enters thespecimen 55 as S-polarized light, but the present invention is notlimited to this mode, and the modes of rotation of the ½ wavelengthplate 63 and the diffraction grating 64 can be freely set, e.g. rotatingthe ½ wavelength plate 63 and the diffraction grating 64 in oppositedirections, only if the illumination light generated by the diffractiongrating 64 results in S-polarized light.

In concrete terms, in the case of FIG. 12B, the beam splitting directionof the diffraction grating 64 is in a position that is rotated to theright from the fourth reference position by the rotation angle α, andthe position of the fast axis of the ½ wavelength plate 63, in order togenerate a polarization state such that the pair of ±first orderdiffracted lights generated by the diffraction grating 64 is irradiatedonto the specimen 55 as S-polarized light, is not limited to a positionthat is rotated to the right from the third reference position by therotation angle α/2.

In other words, the position that is rotated from this position by ±90°(that is, the position that is rotated to the left from the thirdreference position by 90°−α/2, which results in the same position as theposition that is rotated to the right from the third reference positionby α/2+90°) is also a position where the same polarization state isgenerated. Therefore in the case of rotating the beam splittingdirection of the diffraction grating 64 to the right from the fourthreference position by the rotation angle α, the same functional effectcan be implemented by rotating the fast axis of the ½ wavelength plate63 to the left from the third reference position by 90°−α/2, forexample, hence this latter mode may be used instead. The third referenceposition and the fourth reference position mentioned above are notlimited to the positions indicated in FIG. 12, but may be set at anyappropriate position.

Embodiment 4

In Embodiment 1 described above, the direction of the fast axis of the ½wavelength plate 17 is set using a configuration that allows the ½wavelength plate 17 to rotate around the optical axis of the ½wavelength plate 17, but the direction of the fast axis may be set bydisposing at least two liquid crystal elements (first liquid crystalelement 101 and second liquid crystal element 102 in order from thelight source) as illustrated in FIG. 14A, instead of the ½ wavelengthplate 17 in FIG. 1, and allowing the liquid crystal elements to functionas the ½ wavelength plate according to the following control.

The axial direction of the polarizing plate 23 is set such that thepolarization direction of incoming beams with respect to the diffractiongrating 13 is the same as the direction of the grid line (marked line)for the splitting direction V₁ of the diffraction grating 13 indicatedin FIG. 2A. Since the first liquid crystal element 101 is an elementwhich can control refractive index anisotropy by electricallycontrolling the liquid crystal orientation, the first liquid crystalelement 101 functions as a ½ wavelength plate for the incoming beams ifturned ON by the driving circuit 101A, and functions as a plane parallelplate (refractive index anisotropy is 0, refractive index is isotropic)for the incoming beams if turned OFF by the driving circuit 101A. Thedirection of the fast axis of the first liquid crystal element 101 inthe ON state is a −30° rotated direction from the axial direction of thepolarizing plate 23 (+ is the clockwise direction).

The structure of the second liquid crystal element 102 is the same asthe structure of the first liquid crystal element 101, and therefore thesecond liquid crystal element 102 functions as the ½ wavelength platefor the incoming light when turned ON by the driving circuit 102A, andfunctions as a plane parallel plate for the incoming light if turned OFFby the driving circuit 102A. The direction of the fast axis of thesecond liquid crystal element 102 in the ON state however is differentfrom the direction of the fast axis of the first liquid crystal element101, and is set to a +30° rotated direction from the axial direction ofthe polarizing plate 23.

As illustrated in FIG. 14B, if ±first order diffracted light beams thatare split in the direction V₁ at 0° (a pair of collected points 14 g and14 d arranged in the direction V₁ at 0°), ±first order diffracted lightbeams that are split in the direction V₂ at 120° (a pair of collectedpoints 14 c and 14 f arranged in the direction V₂ at 120°), and ±firstorder diffracted light beams that are split in the direction V₃ at 240°(a pair of collected points 14 b and 14 e arranged in the direction V₃at 240°) must be S-polarized when irradiated onto the specimen 5respectively, that are the polarization direction of each light afterpassing through the ½ wavelength plate (first liquid crystal element 101or second liquid crystal element 102) must be in the direction indicatedby the arrows in FIG. 14B respectively.

Therefore in the case of the ±first order diffracted light beams splitin the direction V₂ at 120°, the direction of the fast axis of the ½wavelength plate (first liquid crystal element 101) must be set to adirection that is rotated from the axial direction of the polarizingplate 23 by −30° (−30° from the grid line to indicate the splittingdirection V₁ in FIG. 2A), that is, ½ of the angle formed by thepolarization direction of the light after passing through the ½wavelength plate (−60° from the grid line to indicate the splittingdirection V₁ in FIG. 2A) and the polarization direction of the lightbefore passing through the ½ wavelength plate (the same direction as thegrid line to indicate the splitting direction V₁ in FIG. 2A).

In the case of the ±first order diffracted light beams that are split inthe direction V₃ at 240°, the direction of the fast axis of the ½wavelength plate (second liquid crystal element 102) must be set to adirection that is rotated from the axial direction of the polarizingplate 23 by +30° (+30° from the grid line to indicate the splittingdirection V₁ in FIG. 2A), that is, ½ of the angle formed by thepolarization direction of the light after passing through the ½wavelength plate (+60° from the grid line to indicate the splittingdirection V₁ in FIG. 2A), and the polarization direction of the lightbefore passing through the ½ wavelength plate (the same direction as thegrid line to indicate the splitting direction V₁ in FIG. 2A).

Therefore the polarization direction of the beam which has passedthrough the first liquid crystal element 101 and the second liquidcrystal element 102 becomes the same direction as the grid line toindicate the splitting direction V₁ in FIG. 2A in a state where both thefirst liquid crystal element 101 and the second liquid crystal element102 are turned OFF, becomes the same direction as the grid line toindicate the split direction V₂ in FIG. 2A in a state where only theliquid crystal element 101 is turned ON, and becomes the same directionas the grid line to indicate the splitting direction V₃ in FIG. 2A in astate where only the liquid crystal element 102 is turned ON.

If the pair of ±first order diffracted lights 14 g and 14 d whichbelongs to the first group of beams is selected out of the three pairsof ±first order diffracted lights, as illustrated in FIG. 4A, thedirection of the fast axis of the ½ wavelength plate 17 must be set to adirection that is rotated from the axis of the polarizing plate 23 by+θ₁/2, that is ½ of the angle formed by the polarization direction ofthe light after passing through the ½ wavelength plate 17 (+θ₁ from theaxis of the polarizing plate 23) and the polarization direction of thelight before passing through the ½ wavelength plate 17 (axial directionof the polarizing plate 23).

In this case, the polarization direction of each ±first order diffractedlight before passing through the ½ wavelength plate 17 (indicated by thebroken line with bidirectional arrows in FIG. 4A) is parallel with theaxial direction of the polarizing plate 23, but the polarizationdirection of each ±first order diffracted light after passing throughthe ½ wavelength plate 17 rotates to the right by the rotation angle θ₁(in FIG. 4A, only the polarization directions of the pair of ±firstorder diffracted lights 14 g and 14 d selected by the beam selectionmember 18 are indicated by the solid line with bidirectional arrows),and becomes perpendicular to the beam selection direction of the beamselection member 18 (matches with the split direction V₁ indicated inFIG. 2A).

In the same manner, if the pair of ±first order diffracted lights 14 cand 14 f which belongs to the second group of beams is selected out ofthe three pairs of ±first order diffracted lights, as illustrated inFIG. 4B, the direction of the fast axis of the ½ wavelength plate 17must be set to a direction that is rotated from the axis of thepolarizing plate 23 by +θ₂/2, that is ½ of the angle formed by thepolarization direction of the light after passing through the ½wavelength plate 17 (+θ₂ from the axis of the polarizing plate 23) andthe polarization direction of the light before passing through the ½wavelength plate 17 (axial direction of the polarizing plate 23).

In this case, the polarization direction of each ±first order diffractedlight before passing through the ½ wavelength plate 17 (indicated by thebroken line with bidirectional arrows in FIG. 4B) is parallel with theaxial direction of the polarizing plate 23, but the polarizationdirection of each ±first order diffracted light after passing throughthe ½ wavelength plate 17 rotates to the right by the rotation angle θ₂(in FIG. 4B, only the polarization directions of the pair of ±firstorder diffracted lights 14 c and 14 f selected by the beam selectionmember 18 are indicated by the solid line with bidirectional arrows),and becomes perpendicular to the beam selection direction of the beamselection member 18 (matches with the splitting direction V₂ indicatedin FIG. 2A).

Further, if the pair of ±first order diffracted lights 14 b and 14 ewhich belongs to the third group of beams is selected out of the threepairs of ±first order diffracted light, as illustrated in FIG. 4C, thedirection of the fast axis of the ½ wavelength plate 17 must be set to adirection that is rotated from the axis of the polarizing plate 23 by−θ₁/2, that is, ½ of the angle formed by the polarization direction ofthe light after passing through the ½ wavelength plate 17 (−θ₃ from theaxis of the polarizing plate 23) and the polarization direction of thelight before passing through the ½ wavelength plate 17 (axial directionof the polarizing plate 23).

In this case, the polarization direction of each ±first order diffractedlight before passing through the ½ wavelength plate 17 (indicated by thebroken line with bidirectional arrows in FIG. 4C) is parallel with theaxial direction of the polarizing plate 23, but the polarizationdirection of each ±first order diffracted light after passing throughthe ½ wavelength plate 17 rotates to the left by the rotation angle θ₃(in FIG. 4C, only the polarization directions of the pair of ±firstorder diffracted lights 14 b and 14 e selected by the beam selectionmember 18 are indicated by the solid line with bidirectional arrows),and becomes perpendicular to the beam selection direction of the beamselection member 18 (matches with the splitting direction V₃ indicatedin FIG. 2A).

Instead of the ½ wavelength plate 63 in FIG. 10, at least two liquidcrystal elements (first liquid crystal element 101 and second liquidcrystal element 102 in order from the light source), as illustrated inFIG. 14A, may be disposed and allowed to function as the ½ wavelengthplate to set the direction of the fast axis according to the abovementioned control.

As described above, according to the structured illumination microscopedevices in Embodiment 1 to Embodiment 4 of the present invention, mostof the light from the light source can be used as S-polarized structuredillumination light, therefore light utilization efficiency can beenhanced.

In Embodiment 1 to Embodiment 4, the direction of the fast axis of the ½wavelength plate 17 is described based on the assumption that the angleformed by the polarization direction of the light after passing throughthe ½ wavelength plate 17 and the polarization direction of the lightbefore passing through the ½ wavelength plate 17 (axial direction of thepolarizing plate 23) is an acute angle, but may be an obtuse angle(S-polarized light of which phase is different by a value of π).

In the above description, the rotation angle of the ½ wavelength plate17 with respect to the beam selection direction of the beam selectionmember 18 or 18A, and the rotation angle of the ½ wavelength plate 63with respect to the beam splitting direction of the diffraction grating64, are accurately specified, but an error in the ±5° ˜6° range isallowed.

It is preferable to use the ½ wavelength plate 17 and the ½ wavelengthplate 63 corresponding to the wavelength of the light source, since thephase difference shifts from the original phase difference of the ½wavelength plate if the wavelength of the light source changes, but awide-band wavelength plate of which change of phase with respect to the½ wavelength is small when the wavelength is wide may be used. Even inthis case, it is preferable that the phase difference between anordinary ray and an extraordinary ray is within ±5°. If the phasedifference between an ordinary ray and an extraordinary ray is within±5°, the light intensity change when linearly polarized light isextracted can be kept within 1%.

In Embodiment 1 and Embodiment 2, the rotation of the beam selectionmember 18 or 18A and the ½ wavelength plate 17 is stopped when aplurality of phase-modulated images is captured, but phase may bemodulated while continuously rotating the beam selection member 18 or18A and the ½ wavelength plate 17, and an image may be captured eachtime phase is modulated.

In Embodiment 1 and Embodiment 2, the beam selection member 18 or 18Acan select only a beam in one direction at a time out of the beams(diffracted light) split into three directions by the diffractiongrating 13, but a plurality of beams in mutually different directionsmay also be selected at one time. In this case, in the structuredillumination light to be generated, fringes in a plurality of differentdirections are superposed.

The function of the ½ wavelength plate used in the present invention (togenerate linearly polarized light in an arbitrary direction) may beimplemented using two ¼ wavelength plates, but the present inventionthat uses one ½ wavelength plate has the following advantages over thecase of using two ¼ wavelength plates, in addition to cost reduction dueto the use of less number of components.

A reflection surface of the optical element on the optical pathdecreases when one ½ wavelength plate is used, compared with the case ofusing two ¼ wavelength plates, hence the influence of unwanted reflectedlight can be decreased. Further, the influence of an error of thewavelength characteristics of each wavelength plate can be decreasedwhen one ½ wavelength plate is used, compared with the case of using two¼ wavelength plates, hence the use of one ½ wavelength plate isparticularly advantageous when a wide-band light is used.

In each of the above mentioned embodiments, the diffraction grating isused as the beam splitting unit, but another optical element, such as aprism or a mask which can rotate or linearly move and on which periodicpatterns (a plurality of periodic patterns having mutually differentdirections of a repetition cycle) are formed, may be used as the beamsplitting unit as long as this optical element does not interruptcoherence of the light.

In this case, the fast axis of the ½ wavelength plate is controlled(rotated) so as to be maintained at a ½ position of an angle formed bythe polarization direction of the beam that enters the ½ wavelengthplate and the polarization direction of the beam that is emitted fromthe ½ wavelength plate, according to the polarization direction of thebeam emitted from the ½ wavelength plate, which is determined dependingon the direction perpendicular to the direction of the repetition cyclethat rotates or linearly moves according to the rotation or linearmovement.

In Embodiment 1, Embodiment 2 and Embodiment 4, the ½ wavelength plate17 is disposed before (the light source side of) the beam selectionmember 18 or 18A, but the ½ wavelength plate 17 may be disposed after(the microscope side of) the beam selection member 18 or 18A. In thesame manner, in Embodiment 3, the ½ wavelength plate 63 is disposedbefore (the light source side of) the diffraction grating 64, but the ½wavelength plate 63 may be disposed after (the microscope side of) thediffraction grating 64.

The present invention can be applied not only to a fluorescentobservation device, but also to various observation devices(microscopes) which perform structured illumination.

In this description, the following disclosed documents are incorporatedby reference.

-   1) U.S. Pat. No. 6,239,909-   2) U.S. Reissue Pat. No. 38307-   3) U.S. Pat. No. 8,115,806

1. A structured illumination optical system comprising: a beam splitterdisposed near a conjugate position of a specimen and configured to splitbeams from a light source into a plurality of groups of beams havingdifferent splitting directions around a predetermined axis; a beamselector configured to select one group of beams from the plurality ofgroups of beams; and a ½ wavelength plate, wherein the structuredillumination optical system is configured to form, on the specimen,interference fringes by a plurality of beams included in the one groupof beams, and a fast axis of the ½ wavelength plate is set, based on theone group of beams selected by the beam selector, to a directiondisplaced from a predetermined direction by an angle θ/2, where θ is adifference between (i) a polarization direction of a beam that entersthe ½ wavelength plate and (ii) a polarization direction of a beam thatis to be emitted from the ½ wavelength plate.
 2. The structuredillumination optical system according to claim 1, wherein thepolarization direction of the beam that is to be emitted from the ½wavelength plate is perpendicular to the splitting direction of the onegroup of beams that has been selected by the beam selector and split bythe beam splitter.
 3. The structured illumination optical systemaccording to claim 1, wherein the ½ wavelength plate and the beamselector are rotatable with respect to an optical axis of the structuredillumination optical system, and rotation angles of the 4 wavelengthplate and of the beam selector with respect to the optical axis are setrespectively.
 4. The structured illumination optical system according toclaim 3, wherein the rotation angle of the ½ wavelength plate is ½ ofthe rotation angle of the beam selector.
 5. The structured illuminationoptical system according to claim 3, wherein the ½ wavelength platerotates in a same direction as the beam selector at a rotation speedthat is ½ rotation speed of the beam selector.
 6. The structuredillumination optical system according to claim 1, wherein the ½wavelength plate is a liquid crystal element, and the fast axis is setby controlling driving of the liquid crystal element.
 7. The structuredillumination optical system according to claim 1, wherein the ½wavelength plate is disposed near the beam selector.
 8. A structuredillumination optical system comprising: a beam splitter that is disposednear a conjugate position of a specimen and configured to split a beamfrom a light source into a plurality of beams; a controller configuredto control a splitting direction of the plurality of beams; and a ½wavelength plate, wherein the structured illumination optical system isconfigured to form, on the specimen, interference fringes by theplurality of beams, and a fast axis of the ½ wavelength plate is set,based on the splitting direction of the plurality of beams, to adirection displaced from a predetermined direction by an angle θ/2,where θ is a difference between (i) a polarization direction of a beamthat enters the ½ wavelength plate and (ii) a polarization direction ofa beam that is to be emitted from the ½ wavelength plate.
 9. Thestructured illumination optical system according to claim 8, wherein thepolarization direction of the beam that is to be emitted from the ½wavelength plate is perpendicular to the splitting direction of theplurality of beams.
 10. The structured illumination optical systemaccording to claim 8, wherein rotation of the ½ wavelength plate androtation of the beam splitter are controlled with respect to an opticalaxis of the structured illumination optical system by the controller,and rotation angles of ½ the wavelength plate and of the beam splitterwith respect to the optical axis are set respectively.
 11. Thestructured illumination optical system according to claim 10, whereinthe rotation angle of the ½ wavelength plate is ½ of the rotation angleof the beam splitter.
 12. The structured illumination optical systemaccording to claim 10, wherein the ½ wavelength plate rotates in a samedirection as the beam splitter at a rotation speed that is ½ a rotationspeed of the beam splitter.
 13. The structured illumination opticalsystem according to claim 8, wherein the ½ wavelength plate is a liquidcrystal element, and the fast axis is set by controlling driving of theliquid crystal element.
 14. The structured illumination optical systemaccording to claim 8, wherein the ½ wavelength plate is disposed nearthe beam splitter.
 15. The structured illumination optical systemaccording to claim 1, wherein a phase difference between an ordinary rayand an extraordinary ray of the beam in the ½ wavelength plate is within±5°.
 16. The structured illumination optical system according to claim8, wherein a phase difference between an ordinary ray and anextraordinary ray of the beam in the ½ wavelength plate is within ±5°.17. A structured illumination microscope device comprising: thestructured illumination optical system according to claim 1; a phasemodulator configured to modulate a phase of the plurality of beams; animage optical system that forms an image of light from the specimen onan imaging plane of an imaging device; and an image processing unit thatgenerates a specimen image by performing arithmetic processing on aplurality of images imaged by the imaging device each time the phasemodulator modulates the phase of the plurality of beams.
 18. Thestructured illumination microscope device according to claim 17, whereinthe phase modulator modulates the phase of the plurality of beams bydriving the beam splitter in a direction perpendicular to an opticalaxis of the structured illumination optical system.
 19. The structuredillumination microscope device according to claim 17, further comprisinga driving amount determination unit that determines a driving amount ofthe beam splitter by the phase modulator, on the basis of a phasemodulation amount of the plurality of beams.
 20. A structuredillumination microscope device comprising: the structured illuminationoptical system according to claim 8; a phase modulator configured tomodulate a phase of the plurality of beams; an image optical system thatforms an image of light from the specimen on an imaging plane of animaging device; and an image processing unit that generates a specimenimage by performing arithmetic processing on a plurality of imagesimaged by the imaging device each time the phase modulator modulates thephase of the plurality of beams.
 21. The structured illuminationmicroscope device according to claim 20, wherein the phase modulatormodulates the phase of the plurality of beams by driving the beamsplitter in a direction perpendicular to an optical axis of thestructured illumination optical system.
 22. The structured illuminationmicroscope device according to claim 20, further comprising a drivingamount determination unit that determines a driving amount of the beamsplitter by the phase modulator, on the basis of a phase modulationamount of the plurality of beams.